U.S. patent number 4,933,291 [Application Number 07/095,693] was granted by the patent office on 1990-06-12 for centrifugable pipette tip and pipette therefor.
This patent grant is currently assigned to Eastman Kodak Company. Invention is credited to John L. Daiss, Alan J. Lowne, Leonard J. Seaberg.
United States Patent |
4,933,291 |
Daiss , et al. |
June 12, 1990 |
Centrifugable pipette tip and pipette therefor
Abstract
There are described a novel removable pipette tip and pipette
therefor, which provide separation of substances of a liquid within
the tip by centrifuging the tip. The tip comprises a
liquid-confining cavity disposed about an axis of symmetry, a
dispensing aperture, and separating structure spaced along the axis
away from the dispensing aperture, for separating one of the liquid
substances when the tip is spun about such axis. The pipette is
rotatable and either the pipette or the analyzer includes a driver
for spinning the pipette tip at high speed.
Inventors: |
Daiss; John L. (Rochester,
NY), Seaberg; Leonard J. (Penfield, NY), Lowne; Alan
J. (Victor, NY) |
Assignee: |
Eastman Kodak Company
(Rochester, NY)
|
Family
ID: |
26790497 |
Appl.
No.: |
07/095,693 |
Filed: |
September 14, 1987 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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944778 |
Dec 22, 1986 |
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Current U.S.
Class: |
436/45; 356/244;
356/246; 422/513; 422/561; 422/72; 422/931; 436/177; 494/16;
494/17; 494/19; 73/863.23; 73/864.11; 73/864.13; 73/864.21 |
Current CPC
Class: |
B01L
3/0275 (20130101); G01N 2035/00495 (20130101); Y10T
436/111666 (20150115); Y10T 436/25375 (20150115) |
Current International
Class: |
B01L
3/02 (20060101); G01N 35/00 (20060101); G01N
035/00 () |
Field of
Search: |
;422/72,100,101
;436/45,177 ;494/16,17,19 ;356/246,244
;73/863.23,864.11,864.13,864.21 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Richman; Barry S.
Assistant Examiner: Kummert; Lynn M.
Attorney, Agent or Firm: Schmidt; Dana M.
Claims
What is claimed is:
1. A pipette tip capable of separating portions of a liquid
solution, emulsion or dispersion said tip comprising
means for mounting the tip within a pipette,
a body wall disposed about an axis of symmetry to define a
liquid-confining cavity of the tip,
means defining a dispensing aperture in said body wall,
and separating means for separating, and maintaining separate, a
first portion of the liquid solution, emulsion or dispersion from a
second portion when the tip is spun about said axis of
symmetry.
2. A tip as defined in claim 1, wherein said separating means
extends concentrically and entirely around said axis of
symmetry.
3. A tip as defined in claim 1, wherein said separating means
includes a trap cavity and partitioning means for keeping the first
portion contained within said trap cavity, said body wall being
shaped to direct liquid flow towards said trap cavity during
spinning.
4. A tip as defined in claim 3, wherein said partitioning means is
a lip that extends from said body wall, and the first portion is
the denser of two portions of the liquid solution, emulsion or
dispersion.
5. A tip as defined in claim 3, wherein said separating means
includes a filter completely closing off said trap cavity from said
liquid-confining cavity, except for pores in said filter.
6. A tip as defined in claim 5, wherein said filter is constructed
with pore sizes selected to pass liquid through the filter and to
retain particulate or cellular matter contained in the liquid.
7. A tip as defined in claim 5, wherein said filter is chemically
treated to bind the second portion and to allow the first portion
to pass through to said trap cavity, and wherein the second portion
is a selected component of the solution, emulsion or
dispersion.
8. A tip as defined in claim 5, wherein said filter is an
annulus.
9. A pipette tip capable of separating two different-density phases
of a two-phase liquid, said tip comprising
a body wall disposed about an axis of symmetry to define a primary
cavity of the tip, the wall having a varying spacing distance from
said axis along at least a portion of the length of said wall,
means defining a dispensing aperture in said body wall, said
spacing distance of said wall increasing with increased distance
from said aperture,
a trap cavity fluidly connected to said primary cavity and
extending concentrically around said axis, said trap cavity having
a volume less that the volume of said primary cavity but sufficient
to hold substantially all of the denser of said two phases,
said trap cavity being disposed on said body wall spaced along said
axis away from said dispensing aperture,
the portion of said body wall extending between said dispensing
aperture and said trap cavity being shaped to have a slope relative
to said axis that is sufficient to force substantially all of said
denser phase to flow into said trap cavity when said tip is spun
about said axis,
and a partitioning lip extending concentrically around said axis
between said primary cavity and said trap cavity, said lip having a
height measured parallel to said axis sufficient to prevent gravity
flow of collected denser phase out of said trap cavity and into
said primary cavity when said axis is vertical.
10. A pipette tip as defined in claim 9, and further including
means thereon for engaging a high speed spinning means.
11. In a pipette for aspirating and dispensing a liquid and having
a fluid passageway cooperating with a pipette tip, first means for
evacuating and pressurizing said fluid passageway for filling and
dispensing the liquid from the tip, and means for removably
mounting the tip,
the improvement wherein said mounting means is constructed to
permit the tip to rotate continuously at high speed about an axis
of symmetry thereof.
12. A pipette as defined in claim 11, and further including means
for rotating said mounting means or the tip at high speed.
13. In a combination comprising a pipette having an axis and
comprising a fluid passageway and first means for evacuating and
pressurizing said fluid passageway,
and a removable pipette tip connected to said passageway for
collecting and dispensing liquid in response to said first means
for evacuating and pressurizing,
the improvement wherein said pipette further includes means for
mounting said tip to rotate continuously on said pipette about said
axis, and wherein said tip includes means for separating, and
maintaining separate, a portion of the liquid contained therein
centrifugally separated from the remainder in response to spinning
at high speeds about said axis.
14. A combination as defined in claim 13, and further including
means in said pipette for spinning said tip at high speeds about
said axis.
15. In an analyzer comprising a first station constructed to
dispense a body liquid onto a test element using a pipette provided
with a pipette tip, a second read station including means for
detecting a change in the test element in response to the body
liquid, and means for transporting the test element from one of
said stations to the other,
the improvement wherein said first station includes means for
spinning said pipette tip at high speeds about an axis of symmetry
of said tip.
16. A method of separating two portions of a liquid solution,
emulsion or dispersion in a pipette, comprising the steps of
(a) aspirating the liquid solution, emulsion or dispersion into a
tip comprising means for mounting the tip within a pipette, a body
wall disposed about an axis of symmetry to define a
liquid-confining cavity of the tip, means defining a dispensing
aperture in said body wall, and separating means for separating,
and maintaining separate, a first portion of the liquid solution,
emulsion or dispersion from a second portion when the tip is spun
about said axis of symmetry, said tip being mounted on the pipette,
and
(b) spinning the tip to force a portion of the liquid solution,
emulsion or dispersion past said separating means.
Description
FIELD OF THE INVENTION
This invention relates to pipettes used to collect and dispense
liquids, for example, liquids characterized by two phases of
different densities.
BACKGROUND OF THE INVENTION
A large industry has developed around dried test elements used
clinically to analyze serum or plasma for analytes that are a
measure of a patient's health. Generally, such test elements have
been designed exclusively for testing serum or plasma, in light of
the fact that cells from whole blood cause interferences of various
kinds. The drawback of such an approach is the necessity for
centrifuging the patient sample first, to remove the unwanted blood
cells. Conventionally, this has required the use of a separate
centrifuge device and sample containers as well as separate
operator involvement.
Such a centrifuge step has been only a minor inconvenience in those
instances in which the test elements are used in hospitals or large
laboratories. The reason is that such institutions have the
equipment and expertise to readily perform the centrifuge
separation step. However, the test elements and an appropriate
analyzer have recently moved into the environment of the doctors'
office. There, the need for a separate centrifuge step is a major
drawback, since many doctors' offices lack the equipment and
training to routinely do centrifuging prior to testing.
Furthermore, the centrifuging step is time-consuming. Rapid testing
is the essence of tests run in the doctors' office, in order to
complete the diagnosis while the patient is still present.
Attempts have been made to convert so-called dried test elements
used to analyze serum or plasma, into test elements useful also to
test whole blood. Such attempts have featured the addition of a
blood-cell filtering layer, above the spreading layer heretofore
constituting the outermost layer. The purpose is to cause the cells
to separate from the plasma, the cells being retained within the
filter layer. In this way, the centrifuging step heretofore needed
to obtain just serum or plasma from the whole blood, is eliminated.
Examples are shown in EPO Application No. 0,159,727.
However, there are drawbacks to the approach using a blood-cell
filtering layer. Chief of these is that there does not appear to be
a single filter material that works for all the various test
chemistries needed for the many different analytes. This may be
partly due to the fact that some assays need to have reagents in
the spreading layer (heretofore the outermost layer), and some have
no reagents there. As a result, it has been difficult to obtain
whole blood test elements for all the analytes currently tested in
serum or plasma.
Therefore, prior to this invention there has been a need to provide
test elements and analyzers that allow the direct testing of all
analytes of whole blood, without requiring a preliminary blood cell
separation step that involves separate equipment and operator
involvement. Thus there is an important need, particularly in the
doctors' office, is to provide a whole blood clinical analyzer for
all analytes that is largely user transparent to the fact that some
kind of cell-plasma separation step occurs during the process. (As
used herein, "user transparent" means that the user involvement in
achieving the noted step is minimal or non-existent.)
SUMMARY OF THE INVENTION
We have devised a pipette construction which provides for automatic
separation of liquid components of a solution, dispersion or
emulsion, such as cells from plasma, almost immediately upon
collection of whole blood in the pipette tip. The features which
make this possible are a novel pipette tip, a novel pipette, and
optionally, a novel analyzer.
More specifically, in accord with one aspect of the invention there
is provided a pipette tip capable of separating portions of a
liquid solution, emulsion or dispersion, the tip comprising
means for mounting the tip within a pipette,
a body wall disposed about an axis of symmetry to define a
liquid-confining cavity of the tip,
means defining a dispensing aperture in the body wall,
and separating means for separating, and maintaining separate, a
first portion of the liquid solution, emulsion or dispersion from a
second portion when the tip is spun about the axis.
In accord with another aspect of the invention, there is provided a
pipette for aspirating and dispensing a liquid and having a fluid
passageway cooperating with a pipette tip, first means for
evacuating and pressurizing the fluid passageway for filling and
dispensing such liquid from the tip, and means for removably
mounting such tips. This pipette is improved in that the mounting
means is constructed to permit the tips to rotate repeatedly at
high speed about an axis of symmetry.
In accord with still another aspect of the invention, a combination
of pipette and removable pipette tip is provided, the pipette
having the axis, fluid passageway and first means described in the
previous paragraph. This combination is improved in that the
pipette further includes means for mounting the tip to rotate
continuously on the pipette about the axis, and wherein the tip
includes means for separating, and for maintaining separate, a
portion of the liquid contained therein from the remainder in
response to spinning at high speeds about the axis.
In accord with yet another aspect of the invention, there is
optionally provided an analyzer comprising a first station
constructed to dispense a body liquid onto a test element using a
pipette provided with a pipette tip, a second read station
including means for detecting a change in such test element in
response to such body liquid, and means for transporting such test
element from one of the stations to the other. The analyzer is
improved in that the first station includes means for spinning the
pipette tip at high speeds about an axis for symmetry.
Thus, it is an advantageous feature of the invention that whole
blood is tested for analytes using test elements constructed for
serum or plasma only, without requiring additional equipment used
only for whole blood separation.
It is a related advantageous feature of the invention that whole
blood is tested for analytes using test elements constructed for
serum or plasma only, by equipment which is generally user
transparent to the fact that a preliminary cell-plasma separation
step takes place.
It is another advantageous feature of the invention that a pipette
is provided that permits ready separation of a two-phase liquid
having different-density phases, for uses other than dispensing
plasma onto test elements.
Another advantageous feature of the invention is that components of
a solution, an emulsion, or a dispersion can be separated by
filtration within a pipette, with the filtered component being
retained in position for subsequent treatment.
Other advantageous features will become apparent upon reference to
the following description of the preferred embodiments, when read
in light of the attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a sectional view, taken along an axis of symmetry, of a
pipette tip constructed in accordance with the invention;
FIG. 2 is a partially schematic, sectional view of a pipette
constructed in accordance with the invention, for use with the tip
of FIG. 1;
FIG. 2A is a fragmentary sectional view similar to a portion of
FIG. 2, but of an alternative embodiment;
FIGS. 3A-3D are fragmentary sectional views similar to that of FIG.
1, illustrating one manner of use of the pipette and pipette
tip;
FIG. 4 is a partially schematic, fragmentary sectional view of an
improved analyzer constructed in accordance with the invention;
FIG. 5 is a sectional view similar to that of FIG. 1, but of an
alternate embodiment.
FIG. 6 is a sectional view similar to that of FIG. 1, but
illustrating still another embodiment;
FIG. 7 is a section view similar to that of FIG. 6, illustrating a
preferred use;
FIG. 8 is a fragmentary section view of a tip similar to that of
FIG. 6, except yet another embodiment is illustrated; and
FIG. 9 is a section view similar to FIG. 1, but of still another
alternate embodiment.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The invention is described hereinafter primarily for use in
separating portions of a liquid from each other, for example, blood
cells from plasma, a preferred embodiment, or substances in a
liquid solution, emulsion or dispersion from the rest of the
liquid, e.g., from the solvent. That is, whole blood is the
preferred two-phase liquid processed by this invention, the denser
phase being blood cells and platelets. In addition, the invention
is useful in processing other liquids, regardless of the end use
that is made of the separated portion. For example, the liquid can
be a dispersion of yeast cells, and the desired less dense phase be
cell-free culture supernatant. Yet another example is applicable to
immunoassays. In some applications, competitive binding allows some
labeled antigen to compete with unlabeled patient antigen for sites
on an appropriate antibody. After the competitive binding is
complete, dextran-coated charcoal is added to the solution, and the
mixture is aspirated into the pipette. Following centrifugation
within the tip of the pipette, the bound antibody-antigen complex,
which is not capable of absorbing onto the charcoal, is the
separated supernatant and is dispensed to allow measurement of the
amount of bound, labeled antigen.
Yet another preferred use is the separation of cellular components
from urine, blood or liquified tissues so that the cells can be
lysed or otherwise broken down and its contents, such as DNA,
released and processed.
A pipette tip 10 prepared in accordance with the invention
comprises, FIG. 1, a body wall 12 having an axis of symmetry 14,
shaped to define a primary or liquid-confining cavity 16. At one
end of the tip, wall 12 is provided with an aspirating and
dispensing aperture 18 generally centered on axis 14. Wall 12
preferably has a spacing distance "d" from axis 14 that varies
along at least a portion of its length (along axis 14). Most
preferably, wall 12 is shaped so that "d" increases continuously
from its value at aperture 18, to the end of wall 12 forming lip 26
described below, to insure none of the first portion of the liquid,
here the dense phase material, gets stuck in cavity 16 during
centrifuging, but rather moves into annular trap cavity 20. (The
actual angle alpha made by wall 12 against axis 14 can vary widely.
A preferred value is about 20.degree..)
To trap the denser phase during and after centrifuging, annular
cavity 20 is wrapped concentrically around axis 14. There is also
provided partitioning means, preferably a lip 26, that is an
extension of wall 12, for preventing the contents of cavity 20, the
denser phase formed by centrifugal force during spinning, from
moving into aperture 18. Cavity 20 fluidly connects to cavity 16,
by reason of the fact that partitioning lip 26 does not completely
close off cavity 20. As used herein, "fluidly connects" means a
connection that permits ready passage of a liquid between the two
sections or compartments. Specifically, spacing "s" is from about
250 .mu.m (0.010") to about 500 .mu.m (0.020"), and most preferably
about 380 .mu.m (0.015"). Cavity 20 has a volume sufficient to hold
all of the denser phase. The exact amount differs, of course,
depending on the planned use of the tip. For trapping blood cells
of a volume of 200 .mu.l of whole blood contained in cavity 16, the
trap volume is about 100 .mu. l, to accommodate "worst hematocrit"
cases.
Most preferably, lip 26 is provided at the end of wall 12 opposite
to the end with aperture 18. Like cavity 20, it preferably extends
circumferentially completely around axis 14. Its height "h" is
sufficient to retain the denser, trapped phase against flow back
into cavity 16 under the influence of gravity when axis 14 is
vertical. The exact value of h will vary, depending on the volume
of cavity 20. Preferably, h has a value of between about 1 to about
2.0 mm, and most preferably about 1.5 mm, when used with 200 .mu.l
of whole blood.
In a highly preferred example, the total height of cavity 16, from
aperture 18 to the top of lip 26, is about 1.2 cm.
The top of cavity 20 is formed by top wall 30, which has an air
passage 32 extending out therethrough at axis 14. Preferably, a
recess 34 is formed concentrically surrounding passage 32, so as to
create a sharp edge 36. Edge 36 acts to keep separated less dense
phase from creeping along wall 30 back onto recess 34 so as to
retard drainage down into the bottom of cavity 16 after rotation
stops. In addition wall 30 is preferably rounded at portion 38, to
eliminate sharp corners that could prevent the denser phase from
flowing smoothly into cavity 20 during centrifuging.
Nipple 40 extends out the top of wall 30, concentrically around
passage 32, to permit ready mounting of the tip in a pipette.
To render tip 10 readily moldable out of plastics, preferably wall
30 is formed as a separate member 42 with an annular groove 44 near
its outermost circumference. A mating annular lip 46 is formed in
the remaining part of the tip, to mate with groove 44 and form a
leak-tight seal.
Tip 10 can be used with any pipette, including manual pipettes. To
achieve separation within the tip, the pipette preferably mounts
nipple 40 in an air-tight mount, and permits tip 10 to rotate
continuously at high speed.
A preferred form of such a pipette 50 is illustrated in FIG. 2.
Such a pipette comprises a frame 52, a handle portion 54 of the
frame, push-button controls 56, a display 58, a pressurizing and
evacuating chamber 60, a piston 62 movable within the chamber to
create a pressure or a partial vacuum, respectively; motor means 64
for actuating the piston, for example, one available from Airpax
Corp., owned by North American Philips Company, under the trade
name "Airpax Linear Actuator L92100", an air passage 66 extending
from chamber 60, a passageway 68 extending from passage 66, and
means 70 for disconnecting passage 66 from passageway 68, for
example, a disconnect lever 72 actuated by pull rod 74 against a
return spring 76. Rod 74 in turn bears on the end of lead screw 75,
that travels the same direction and distance as piston 62. When rod
74 is released, spring 76 contracts to pull end 77 of passage 66
into sealed engagement with the end of passageway 68. Additionally,
a microprocessor, not shown, is included to control the functions
of the pipette, utilizing power from a line 78 extending out an
umbilical cord 80.
All of the preceding are individually conventional, and require no
further description.
To mount tip 10 on the pipette, a chuck 84 is provided, centered on
axis 14 which coincides with the long axis of the pipette. Chuck 84
is in turn integrally connected to passageway 68 that fluidly
connects passageway 32 of tip 10, FIG. 1, with passage 66 of the
pipette. To permit chuck 84 to rotate continuously, tubular
passageway 68, and thus the chuck, are rotatably mounted withing
bearings 86 and 88. To provide such rotation at speeds of
preferably from 30,000 to 100,000 RPM, an electric motor or air
turbine 90 is mounted on passageway 68. The air turbine is powered
by an air supply, which can be off-pipette and connected to turbine
90 by an air hose 92, or it can be mounted within frame 52. The air
turbine is conventional, and is the type that generates up to
100,000 RPM or more for light-weight loads. Examples can be found
in high-speed dental drills.
Optionally, a tip ejector 100 is also included, comprising a yoke
102 mounted on arm 104. Arm 104 in turn is pivotally attached to
frame 52 at one end 106, and at its other end 108, to an actuating
lever 110. Such lever is manually actuated at handle 54 by means,
not shown.
The mounting of tip within chuck 84 need not be a male-female
connection as shown in FIG. 2. Alternatively, it can be a
female-male configuration as shown in FIG. 2A, wherein nipple 40 is
replaced by a collar 40a. Parts similar to those previously
described bear the same reference numeral, to which the
distinguishing suffix a is applied. Thus collar 40a of tip 10a is
now the female part, and chuck 84a is a male member with passageway
68a extending through it. The advantage of such a construction is
that collar 40a can be more readily used to collect whole blood
directly, as from a finger prick, than can nipple 40.
Alternatively, the pipette can be contructed so that no air
disconnect is needed when spinning is desired. This is
accomplished, not shown, by mounting the dispensing motor so that
its output is connected to a DC motor used to spin the chuck. That
is, the DC motor advances and withdraws, as demanded by the
dispensing motor. The drive shaft of the DC motor in turn is
attached to the piston of a piston chamber, and the piston is
splined to that chamber. The chamber is integrally connected to the
chuck. Thus, when the DC motor is advanced per the dispensing
motor, it pushes the piston relative to the piston chamber, which
cannot reciprocate. But when the DC motor spins its drive shaft,
the entire assembly of the piston and chamber spins, as does the
chuck. (The bearings in this case encase the piston chamber and
piston as well.)
The manner of use of the pipette tip and pipette will be readily
apparent from the preceding discussion. As a further aid in
understanding, reference is made to FIGS. 3A-3D. To aspirate into
the tip the liquid with the two phases, e.g., whole blood, end 77
of the passage 66 is allowed to contact passageway 68, and thus
make fluid contact with the interior of tip 10. In FIG. 3A, this is
symbolized by end 77 making direct contact with the pipette tip,
rotatable passageway 68 being omitted in all of FIGS. 3A-3D for
clarity. Piston 62 is then withdrawn to created a partial vacuum,
as indicated by arrow 111, and the liquid is drawn up to
substantially fill, but not overfill, cavity 16. If what is
aspirated is whole blood from a finger prick, it is preferable that
an anti-coagulant be pre-coated on the inside of the tip. At this
stage, rod 74 is forced against spring 76 by screw 75, to pull end
77 away from passageway 68, arrow 112 of FIG. 3B, to permit
passageway 68, chuck 84 (FIG. 2) and tip 10 to be rotated at high
speed to cause the denser phase, shown in cross-hatching, to flow
into cavity 20, leaving the less dense phase alone in cavity 16.
(Before spinning, surface tension retains liquid in cavity 16 from
spilling out of aperture 18). While spinning, the meniscus 120 of
the less dense phase leaves a temporary hollow passageway 123
centered on axis 14 that is free of liquid. In this manner,
cavities 16 and 20 act together as a blood separation compartment,
lip 26 being effective in maintaining separation of the denser
phase from the less dense. Thus, FIG. 3C, when spinning ceases, the
dense phase is confined to cavity 20 and edge 36 retards any
tendency of the less dense phase to migrate across the surface 34.
Thus, the less dense phase collapses to eliminate passageway 123
and to move adjacent aperture 18 where it is ready for dispensing
onto a test element E, as is shown in FIG. 3D. This last step is
accomplished by rod 74 releasing end 77, arrow 126, to allow fluid
connection with the tip interior, so that pressure, shown as arrow
130, generated in pipette chamber 60, FIG. 2, is effective to
dispense fractions of the less dense phase.
After dispensing is completed, the tip is removed using ejector
yoke 102, FIG. 2, and disposed of.
When used to process whole blood, the pipette tip and pipette of
this invention have been found to effectively separate plasma from
cells as follows, for a preferred embodiment:
______________________________________ Spinning RPM of Time (Sec)
Separation ______________________________________ 40 30,000* 23
40,000 15 50,000 10 60,000 8 70,000 6 80,000 5 90,000 4 100,000
______________________________________ *This is the only one of
this table that was actually measured. The remainder were
calculated on the basis of the ratio of the square of the RPM.
It will thus be readily apparent that the centrifuging step is
largely user transparent. That is, the user pushes button 56, FIG.
2, to start the process which begins with centrifuging. This can
take as little as 4 seconds. Pushing the button a second time
cycles the pipette through the dispensing steps, which can be
easily coordinated with movement of test elements through the
analyzer. Waiting 4 seconds to do the second step is considered to
be a transparent involvement of the user. Alternatively, the
process can be entirely automatic after the pushing of button 56
the first time, by using two-way communication between the pipette
and the analyzer, such as via an infra-red or ultrasonic beam
emitted and received by each of the pipette and analyzer using
conventional equipment.
The previous embodiments utilize a high speed electric spinning
means or air supply for spinning that is supplied via the pipette.
Alternatively, such can be supplied by the analyzer, FIG. 4. Such
an analyzer comprises several stations, the essential ones of which
are a liquid dispensing station 210 and a read station 230. Any
convenient means 240 are useful in advancing a test element E' from
one station to the next, e.g., such as those described in U.S. Pat.
No. 4,303,611, issued on 12/1/81.
At station 210, a pipette 50, shown schematically in FIG. 4, is
supported by suitable conventional means, not shown. The first step
in the process of dispensing plasma occurs when whole blood is
aspirated. Thereafter, a conventional motor 250, e.g., a DC motor
that is part of the analyzer at station 210, rapidly rotates a
suitable drive wheel, such as a capstan roller 260, that bears
against, for illustration, the circumference of tip 10, preferably
at its largest portion. Because tip 10 is free because of bearing
88 to rotate continuously and at high speed, separation of the two
phases occurs in the tip in the same manner as described for the
previous embodiments.
A detectable change is then read, after suitable incubation, at
read station 230. Such station can use, e.g., a light source 220
and a detector 280 oriented on 90.degree.-45.degree. axes, as is
conventional.
The partitioning means for maintaining the separation of the two
liquid phases need not be an extension of body wall 12. FIG. 5
illustrates an embodiment in which the partitioning means is a gel
with a specific gravity inbetween that of the two liquid phases
being separated. Parts similar to those previously described bear
the same reference numerals, to which the distinguishing suffix "b"
is appended.
Thus, the configuration of tip 10b is identical to that of FIG. 1,
except that the lip is replaced by gel 26b. This gel can be any
material that is non-toxic to and non-destructive of the liquid
phases, with a specific gravity that is between about 1.03 and 1.06
if the liquid to be separated is whole blood. Useful examples are
described in, e.g., U.S. Pat. No. 4,050,451, and are conventional
for maintaining the separation of the two phases of whole blood
after centrifuging.
During the spinning of tip 10b, gel 26b tends to be displaced out
of trap cavity 20b by the blood cells, so as to flow towards axis
14b. When spinning is finished, it occupies generally the space
between the vertical dotted lines 100 and 110. The less dense phase
(plasma in the case of whole blood) is then positioned between line
110 and axis 14b.
Alternatively, instead of being positioned initially in cavity 20b,
the gel can be disposed along wall 12b as a thin coating that does
not plug up aperture 18b.
When using gel 26b as the partitioning means, there is no need to
provide edge 36b in upper portion 30b, since the gel prevents
streaming of cells across the top. Accordingly, edge 36b is
optionally eliminated (not shown) in this embodiment.
In the embodiments of FIGS. 6-8, a filter is used in place of a lip
or gel, as the means for separating, and maintaining separate, a
substance of the liquid from the rest of the liquid during
spinning. In these embodiments, the separation is not based upon
density of the components. Rather, it is based upon the use of the
filter to separate one component from the rest, either physically
via pore size selection, or chemically via bonding to chemicals on
the filter. Parts similar to those previously described bear the
same reference numeral, to which the distinguishing suffix "c" is
appended.
Thus, FIG. 6, a pipette tip 10c is prepared as in the previous
embodiments, with a body wall 12c extending around an axis of
symmetry 14c, providing the primary liquid-confining cavity 16c. A
trap cavity 20c is also provided, shaped similarly as in the
previous embodiments. Nipple 40c also functions as described
above.
Unlike the previous embodiments, filter 300 is used in lieu of lip
26, FIG. 1, approximately in the same location as such lip. The
filter completely closes off liquid access from cavity 16c to
cavity 20c, except through the pores of the filter. To that end,
the filter is preferably an annulus extending completely around
axis 14c as shown, although other shapes approximating an annulus
are also useful. Any filter material is useful, such material being
selected with a pore size and of a composition suitable for the
intended filtration. Most preferably, the composition provides a
weakly hydrophobic surface at surfaces 302 and/or 304, and a
membrane intrusion resistance that is less than the centrifugal
force developed by the liquid when tip 10c is spun. In addition,
the pore sizes are selected to retain cellular or other materials
contained in the body fluid comprising the liquid. A 10u
Polycarbonate filter manufactured by Nucleopore is a useful example
of such filter composition.
In use, FIG. 7, tip 10c is spun about axis 14c, as indicated and as
described above, causing the liquid to press against filter 300.
Such liquid pressure overcomes the membrane intrusion resistance
and hydrophobicity at surfaces 302 and 304, so that the liquid
filters through into cavity 20c. Most preferably, therefore, cavity
20c is large enough to accommodate, when tip 10c is not spinning,
all of the liquid previously confined in cavity 16c, below the rim
306 of wall 12c where it would otherwise contact filter 300 if
higher in level. Alternatively, the amount of liquid aspirated into
tip 10c prior to spinning is adjusted to ensure that it will all go
into cavity 20c below the rim 306.
As surface 304 dries out, its weakly hydrophobic nature becomes
restored enough to resist any splashing of liquid in cavity 20c,
from penetrating back to surface 302.
What is retained on surface 302 are cellular products and
particulates that are larger than the pores of filter 300. For
example, leukocytes of blood are retained if the filter pore sizes
are no larger than about 8-10 microns.
Thereafter, additional processing liquid is aspirated into cavity
16c, for example a cellular lysis liquid such as a solution of
quinidinium isothiocyanate, to react with the retained matter on
surface 302. For example, that processing liquid can be used to
extract DNA from leukocytes. The processing liquid is brought into
contact with surface 302 by slowly spinning tip 10c at greatly
reduced speeds. Such reduced speeds are selected so that the
centrifugal force is less than that needed to overcome the membrane
intrusion resistance of filter 300. As a result, contents lysed
from cells trapped at surface 302 are not forced or carried through
the filter to cavity 20c. Instead, they are retained in cavity 16c
for subsequent dispensing.
Thus, tip 10c can be used to separate out particulate or cellular
material from the liquid in a solution, emulsion, or
dispersion.
Alternatively, FIG. 8, the filter can be a composite material.
Parts previously described bear the same reference numeral to which
the distinguishing suffix "d" is appended. Thus, tip 10d is
constructed generally as is described for the embodiment of FIGS. 6
and 7, except that filter 300d is a composite material. More
precisely, there are two annular rings 310 and 312 laminated
together, with ring 310 separating ring 312 from axis 14d. Examples
of ring 310 include chemically treated membranes such as "Nylon
66", and of ring 312, include a woven hydrophobic material such as
"Teflon". Such materials are selected because the pores of ring 310
are best suited to trap and retain the particulate desired, without
necessarily having the necessary resistance to back pressure
generated by the liquid in cavity 20d washing onto surface 304d.
Ring 312, on the other hand, is adapted to resist such
back-washing, without regard to pore sizes particularly needed to
trap the particulates.
Yet another embodiment features the filter and tip construction of
the embodiment of either FIG. 6 or FIG. 8, but wherein a chemical
bond is used, rather than critical pore sizes and physical
separation, to separate a selected component from the liquid
solution, emulsion or dispersion. In such cases a bonding agent is
attached to surface 302 or 302d, to bond directly to the components
to be separated, or to a chemical on such component. Therefore, the
pore sizes of filter 300 or 300d is of no concern in such an
arrangement.
Specific examples of this embodiment include a filter impregnated
with avidin, used to separate out from the rest of the liquid a
biotinylated DNA probe. (The rest of the liquid passes through to
the cavity 20c or 20d.) The bond of the probe to the filter can be
subsequently broken by aspirating into cavity 16c or 16d, a small
quantity of a chaotropic agent used, for example, in affinity
chromatography, and then spinning tip 10c or 10d slowly to cause
wetting of surface 302 or 302d. (Such a technique for breaking such
a bond is described in the prior literature.) Still other examples
are filters coated with polyclonal or monoclonal antibodies that
specifically bind to an antigen of choice, or filters impregnated
with iminobiotin that bond at the imino group with a substance
carrying an avidin group. The antibody-antigen bond, following
separation, can be broken for retrieval of the antigen by treating
surface 302 or 302d, as previously described. The bond between the
iminobiotin and avidin can be subsequently broken, after
separation, by aspirating a buffer such as a mixture of sodium
acetate and acetic acid having a pH of 4, and spinning the tip
slowly to lower the pH of surface 302 or 302d until the bond is
broken, as is well-known.
In some instances, there may be a tendency of the separated lighter
phase to fall out the aperture, once spinning has ceased. In such a
case, the alternate embodiment of FIG. 9 is useful, in which a
ledge 400 is provided adjacent aperture 18e. (Parts similar to
those previously described bear the same reference numeral, to
which the distinguishing suffix "e" is appended. Ledge 400 is
located in a plane that is preferably perpendicular to axis 14e.
That is, the plane of ledge 400 preferably makes an angle to wall
12e that is 90.degree.+.alpha.). Any angle much less than this will
tend to trap air at the pocket formed by the ledge, an undesirable
feature. The remaining features of the tip 10e (16e, 20e, 26e, 30e,
32e, 34e, 38e, 40e, 42e, 44e, 46e) are the same as for the
embodiment of, e.g., FIG. 1.
The invention has been described in detail with particular
reference to preferred embodiments thereof, but it will be
understood that variations and modifications can be effected within
the spirit and scope of the invention.
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